Bound-State Residual Dipolar Couplings for Rapidly Exchanging Ligands of His-Tagged Proteins

Ronald D. Seidel, Tiandi Zhuang, James H. Prestegard
2007 Journal of the American Chemical Society  
The study of bound-state conformations of ligands interacting with proteins is important to the understanding of protein function and the design of drugs that alter function. Traditionally, transferred nuclear Overhauser effects (trNOEs), measured from NMR spectra of ligands in rapid exchange between bound and free states, have been used in these studies, owing to the inherent heavy weighting of bound state data in the averaged ligand signals. In principle, residual dipolar couplings (RDCs)
more » ... ide a useful complement to NOE data in that they provide orientational constraints as opposed to distance constraints, but use in ligand binding applications has been limited due to the absence of heavy weighting of bound-state data. A widely applicable approach to increasing the weighting of bound state data in averaged RDCs measured on ligands is presented. The approach rests on association of a His-tagged protein with a Ni-chelate carrying lipid inserted into the lipidbilayer-like alignment media used in the acquisition of RDCs. The approach is validated through the observation of bound-state RDCs for the disaccharide, lactose, bound to the carbohydrate recognition domain of the mammalian lectin, Galectin-3
doi:10.1021/ja069145h pmid:17385862 pmcid:PMC2542485 fatcat:wxxpzlxhdvdn5kdvdee7fdktfi